Luciferases encompass a wide range of enzymes that catalyze bioluminescence reactions. Bioluminescence is the emission of light produced in a biochemical reaction involving the oxidation of a substrate via an enzyme that occurs within a living organism. Luciferases have been used extensively in different formats for life science research and drug discovery because they are non-toxic, highly sensitive and provide quantitative readouts. Examples of bioluminescence applications include gene reporter assays, whole-cell biosensor measurements, protein-protein interaction studies using bioluminescence resonance energy transfer (BRET), in vivo imaging and drug discovery through high throughput screening (De and Gambhir 2005; Fan and Wood 2007; Ray and Gambhir 2007; Yagi 2007; Sadikot and Blackwell 2008; Prescher and Contag 2010). A number of luciferases have been identified, varying according to their origin, enzyme activity and by their substrate specificity. Of the several luciferases that have been cloned and expressed in bacteria and/or mammalian cells, secreted luciferases have the advantageous characteristic in that their activity can be directly detected in the cell media without disrupting cells (Thompson et al. 1990; Inouye et al. 1992; Maguire et al. 2009; Griesenbach et al. 2011).
Origins of Luciferases
Luciferases are commonly found in lower organisms such as bacteria, fungi, insects, and marine crustaceans. The best-studied luciferases to date are the non-secreted forms of luciferases, such as the firefly luciferase (FLuc) found in the light-emitting organ of the firefly Photinus pyralis (de Wet et al. 1985) and Renilla luciferase (RLuc) from the sea pansy Renilla reniformis (Lorenz et al. 1991). While FLuc and RLuc are widely used as reporters in cultured cells, the major drawback with these non-secreted forms is that they are unsuitable for use in live cells since cell lysis is required prior to measurement of bioluminescence. Less well studied are the naturally secreted forms of luciferases from marine bioluminescent organisms, which carry advantages over non-secreted luciferases for use as reporters to continuously monitor gene expression in live cell-based assays. The secreted luciferase proteins contain signal sequences at their amino-termini targeting the release of the luciferase from the cytosol within the cell to the surrounding culture medium, when expressed either as the wild-type or as a recombinant form in mammalian cells. Thus, genes of secreted luciferases have been exploited for the design of reporter genes, monitoring gene expression in a sample of culture medium without disrupting cells. The first secreted luciferases cloned were Vargula and Cypridina luciferases, isolated from the marine ostracod crustaceans, Vargula hilgendorfi (Thompson et al. 1989) and Cypridina noctiluca (Nakajima et al. 2004), respectively. Their expression in mammalian cells has demonstrated their advantages as secreted reporters for monitoring gene expression in live cells (Inouye et al. 1992; Thompson et al. 1995). Secreted luciferases have since been isolated from the marine copepod crustaceans, Gaussia luciferase (GLuc) from Gaussia princeps (Verhaegent and Christopoulos 2002) and Metridia longa luciferase (MLuc) from Metridia longa (Markova et al. 2004, Golz et al. 2002. Pat. WO 02/42470). Two forms of secreted luciferases, MpLuc1 and MpLuc2, were isolated from another marine copepod Metridia pacifica (Takenaka et al. 2008; Takenaka 2009. U.S. Pat. No. 0,233,320 A1). More recently, in a filed patent (Golz et al. US Pat. No. 2010/0105090), a new secreted luciferase from Metridia longa, named MLuc7 has been described.
Table of LuciferasesNameSpeciesFormSubstrateKineticsFLucPhotinus Non-secretedD-LuciferinGlowpyralis(Firefly)RLucRenillaNon-secretedCoelenterazineFlashreniformisVargulaVargulaSecretedLuciferinGlowhilgendorfiCypridinaCypridinaSecretedLuciferinGlownoctilucaGLucGaussia SecretedCoelenterazineFlashprincepsMLuc/MLuc7Metridia SecretedCoelenterazineFlashlongaMpLuc1/Metridia SecretedCoelenterazineFlashMpLuc2pacificaSubstrates of Luciferases and Enzyme Properties
Substrates of luciferases can be broadly classed into two groups; derivatives of luciferin and coelenterazine. Luciferases using luciferins as substrates require ATP and Mg2+ as cofactors and display stable glow kinetics, whereas luciferases using coelenterazine do not require ATP for activity and, contrary to stable glow kinetics, display rapid flash kinetics. FLuc uses D-luciferin as a substrate and the oxidation reaction emits light with a peak wavelength of 562 nm (de Wet et al. 1985). RLuc uses coelenterazine as a substrate, emitting light with a peak at 480 nm (Lorenz et al. 1991). Although the slightly blue shifted light emission by luciferases that utilize coelenterazine as a substrate is less favorable for their application in in vivo bio-imaging, successful bio-imaging has been described due to their strong bioluminescence signal (De and Gambhir 2005; Griesenbach et al. 2011; Tannous and Teng 2011).
Of the secreted luciferases, Vargula and Cypridina luciferases are similar in size (62 kDa) and display similar enzymatic properties, using Cypridina luciferin as a substrate to produce blue light at a wavelength of 465 nm, oxyluciferin and carbon dioxide. However the secreted luciferases isolated from the marine copepod crustaceans, Gaussia luciferase and Metridia longa luciferase, are considerably smaller proteins employing substrate specificity toward coelenterazine (Verhaegent and Christopoulos 2002; Markova et al. 2004). Gaussia luciferase contains only 185 amino acids (19.9 kDa) while Metridia longa luciferase is a 219-amino acid polypeptide (23.9 kDa). From Metridia pacfica, MpLuc1 consists of 210 amino acids (22.7 kDa) and has the closest homology with Metridia longa luciferase while MpLuc2 (Takenaka et al. 2008) which comprises 189 amino acids (20.3 kDa), has the closest homology with Gaussia luciferase. The recently identified MLuc7 is the smallest luciferase currently known at 169 amino acids (18.4 kDa) (Golz et al. US Pat. No. 2010/0105090).
Similar to the non-secreted RLuc, secreted luciferases from marine copepods catalyze the oxidation of coelenterazine to coelenteramide to produce light at a wavelength of 475 nm, independent of any co-factor (Markova et al. 2004). In summary, distinct properties of the copecod luciferases, primarily the fact that they are secreted and that they display stronger bioluminescence signal render advantages for their use in reporter studies over other luciferases such as FLuc and RLuc (Haugwitz et al. 2008). Furthermore, mutations of secreted luciferases have been described to improved properties such as enhanced light stability and red-shifted emission (Tannous et al. 2005; Haugwitz et al. 2008; Maguire et al. 2009; Welsh et al. 2009; Kim et al. 2011; Tannous and Teng 2011; Tannous et al. 2011, Pat. WO 2011/002924, Kim et al. 2010, Pat. WO 2010/119721).
Applications of Secreted Luciferases
Gaussia luciferase, GLuc, is currently the most exploited secreted luciferase. GLuc cDNA was cloned and overexpressed in a bacterial system, and the protein was purified and used as a sensitive analytical reporter for hybridization assays in vitro (Bryan et al. 2001, U.S. Pat. No. 6,232,107; Verhaegent and Christopoulos 2002). GLuc has also been used for studying a variety of biological processes including quantification of tumor growth (Chung et al. 2009) and monitoring of microbial infections (Enjalbert et al. 2009), as well as in screening for small interfering (si)RNA (Lwa et al. 2010). GLuc is a thermostable, pH resistant protein and in vitro studies have shown that GLuc has more activity than other secreted luciferases as well as the secreted alkaline phosphatase, a commonly used secreted reporter in mammalian cells (Haugwitz et al. 2008). The GLuc cDNA, which consists of 555 by has been humanized by codon optimization for mammalian cell expression. The humanized, codon optimized version of GLuc (hGLuc) was shown to be highly expressed in mammalian cells compared to its wild-type form and to give orders of magnitude stronger bioluminescent signal compared to humanized forms of FLuc and RLuc (Tannous et al. 2005; Maguire et al. 2009). In addition, mutants of GLuc have demonstrated enhanced light stability and red-shifted emission (Maguire et al. 2009; Welsh et al. 2009; Tannous et al. 2011, Pat. WO 2011/002924, Kim et al. 2010, Pat. WO 2010/119721).
In many in vitro and in vivo biological applications, strong and sustained recombinant expression of reporter genes is essential. For instance, reporter genes are playing an increasingly important role in cancer gene therapy as a model transgene, where the aim is to achieve the sustained expression of a variety of anti-tumor proteins such as tumor-suppressor proteins, antigens, cytokines and suicide proteins. The expression stability of a transgene and the protein abundance depends on the nature of the expression cassette. Typically, a codon-optimized gene is combined with a strong viral promoter for enhanced and long-lasting expression. However, such optimized expression cassettes are subject to downregulation of gene expression via transcriptional silencing in vitro and in vivo.
There is a need for a new gene encoding secreted luciferase that displays higher and prolonged gene expression, and generates a secreted luciferase that displays stronger luciferase activity compared to that generated by a codon-optimized luciferase gene such as hGLuc gene.